Chelidonine Induces Caspase-Dependent and Caspase-Independent Cell Death through G2/M Arrest in the T98G Human Glioblastoma Cell Line

Abstract

Chelidonium majus L. (family Papaveraceae), commonly known as greater celandine or tetterwort, has been reported to have antibacterial and anticancer effects and chelidonine is known as a functional metabolite extracted from C.

Figure 1

Chelidonine induces apoptosis in human glioblastoma, T98G cell line. (a) The chemical structure of chelidonine. (b) Human glioblastoma (T98G), lung cancer (A549), breast cancer (MCF7, MDA-MB-231), colon cancer (SW620) cell lines and noncancer (human embryonic kidney cell: HEK293, human umbilical vein endothelial cell: HUVEC, human fibroblast: CCD-25Sk) were treated with chelidonine (1.0 μM) for 24 h, and a dimethylthiazolyl-carboxymethoxyphenyl-sulfophenyl-tetrazolium (MTS) assay was used to determine cell viability. (c) T98G cells were treated with the indicated concentration of chelidonine for 24 h. The size of the sub G_1/0 population of T98G cells, indicative of cell death, was determined by PI staining and flow cytometry analysis. (d) Whole T98G cell lysates were subjected to western blot analysis with the indicated antibodies. Cf; cleaved fragment. (e) Mitochondrial depolarization. Cells were stained with MitoTracker Red CMXRos and then analyzed using flow cytometry. Each experimental result represents the mean ± SEM of three independent experiments. ∗∗∗, p < 0.001, ∗∗, p < 0.01, ∗, p < 0.05 by t-tests.

Figure 2

Chelidonine induces caspase-dependent and -independent apoptosis in T98G cells. (a) Cells were pretreated with 50 μM Z-VAD-FMK for 1 h, followed by treatment with 0.6 μM chelidonine for 24 h. (b) Whole T98G cell lysates were subjected to western blot analysis with the indicated antibodies. The arrows indicate bands corresponding to cleaved caspase-9. Arrow: cleaved caspase-9. (c) T98G cells were synchronized by double thymidine inhibition, washed, and then incubated with 0.6 μM chelidonine for indicated periods of time after synchronization. They were then immunostained for AIF (red) and DNA (DAPI; blue). Images were captured using confocal laser scanning microscopy. Magnification, 600X. Scale bar, 10 μm.

Figure 3

Chelidonine induces G _2/M arrest in T98G cells. T98G cells were seeded in six-well plates and incubated with the indicated concentration of chelidonine for 24 h (a). They were then stained with propidium iodide and analyzed with flow cytometry. (b) The numbers of cells in G_2/M phase of cell cycle were analyzed using ModFit LT™. (c) T98G cells were treated with 2 mM thymidine for 12 h, the thymidine was removed by washing with PBS (3 times), and fresh media was added to the culture plates for 12 h, after which they were retreated with 2 mM thymidine for 12 h. The G_1/0/ arrested cells were then released by PBS washing and the addition of fresh medium containing 0.6 μM chelidonine or DMSO for the indicated periods of time. The cell cycle was analyzed at the indicated time points by PI staining and flow cytometry. The data show the percentages of cells in and G_2/M phase (c) and sub-G_1/0 (d). Error bars represent the standard deviation. The data were analyzed using t-test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 4

Chelidonine-mediated G _2/M arrest induces apoptosis in T98G cells. (a) Chelidonine-mediated multipolar spindle assembly formation. T98G cells were synchronized at G_1/0 through double thymidine inhibition. After the synchronization, the cells were released and cultured in the presence or absence of 0.6 μM chelidonine for 12 or 18 h. Cells were immunostained for α-pericentrin (green), α-tubulin (red), and DNA (DAPI; blue). Images were captured using confocal laser scanning microscope. Magnification, 600×. (b) Whole-cell T98G lysates were subjected to western blot analysis with antibodies against cyclin B1, total or phosphorylated CDK1 (Tyr15 and Thr161), aurora A, total and phosphorylated PLK-1 (Thr210). Tubulin served as a loading control. (c) After the synchronization, cells were cultured in the presence or absence of 10 μM RO-3306 and/or 0.6 μM chelidonine for 24 h. And then the whole-cell T98G lysates were subjected to western blot analysis with the indicated antibodies. (d and e) After the synchronization, cells were cultured in the presence or absence of 10 μM RO-3306 and/or 0.6 μM chelidonine for 24 h. And then the mitochondrial potential (d) and the size of sub G_1/0 population (e) of T98G cells were determined by flow cytometry analysis. Each experimental result is shown as the mean and SEM of three independent experiments. The data was analyzed using t-test. ∗p < 0.05, ∗∗p < 0.01.